Apparatus and method for growing cells

ABSTRACT

A cell-culture apparatus forms an extra-capillary space between at least one hollow fiber and an enclosed chamber. Cells are placed in the extra-capillary space to grow. A media reservoir holds a cell-culture medium. The cell-culture medium is allowed to pass through a lumen of the at least one hollow fiber and to pass nutrients through the walls of the at least one hollow fiber to the cells in the extra-capillary space. Flow through the at least one hollow fiber is produced by action of gravity when a rocking or rotating motion is imparted to the media reservoir.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No.09/885,920, filed Jun. 22, 2001.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention described herein relates to an apparatus and amethod for growing suspension and adherent cells in vitro.

[0004] 2. Background Art

[0005] Growing living cells in vitro is performed for a variety ofpurposes, including the production of cell derivatives, the preparationof viral vaccines, and the recovery of valuable cell by-products. Amongthe devices that have been developed for growing cells in vitro, theshell-and-tube type arrangement has become fairly common, particularlyfor growing suspension and adherent cells.

[0006] These devices use semipermeable tube-shaped hollow fibers (i.e.,capillaries), contained within an outer shell, and configured so thatfluid within a space external to the hollow fibers (i.e., anextra-capillary space) is segregated from fluid passing through thehollow fibers and their corresponding openings (i.e., lumens).Additionally, these devices usually include two manifold end chamberswithin the outer shell on opposite ends of the device. Each of the twolumens of a hollow fiber connects to a different end chamber. The endchambers and the extra-capillary space are separated by thesemi-permeable membranes of the hollow fibers. The composition of theextra-capillary space can be controlled, to a certain extent, by themolecular weight cutoff, or pore size, of the membranes of the hollowfibers.

[0007] Typically, cells are grown in the extra-capillary space while anutrient media is passed through the hollow fibers. The semipermeablenature of the hollow fibers allows nutrients and cell waste products topass through the walls of the hollow fibers while blocking cells fromdoing the same. U.S. Pat. No. 4,391,912 to Yoshida et al. specifies arange of pore diameters to support the transfer of the nutrient mediumfrom the intra-capillary to the extra-capillary space while blocking theentrance of cells into the intra-capillary space.

[0008] Shell-and-tube type bioreactors provide several advantages. Foradherent cells, the use of several hollow fibers provides, within arelatively small volume, a large amount of surface area upon which thecells can grow. For both suspension and adherent cells, this largeamount of surface area also facilitates localized distribution ofnutrient media to the growing cells and ready collection of cell wasteproducts. Shell-and-tube type bioreactors enable the growth of cells atmuch higher density rates than is possible with other cell culturedevices. They can support cell densities greater than 10⁸ cells permilliliter, whereas other cell culture devices are typically limited todensities around 10⁶ cells per milliliter.

[0009] However, existing designs typically require external supportsystems to circulate the nutrient media through the hollow fibers. U.S.Pat. No. 3,883,393 to Knazek et al., U.S. Pat. No. 4,144,136 to Corbeil,U.S. Pat. No. 4,391,912 to Yoshida et al., U.S. Pat. No. 5,290,700 toBinot et al., and U.S. Pat. No. 5,955,353 to Amiot, all teach systems inwhich the nutrient medium is supplied to the intra-capillary spacesusing pumps and associated connection tubing. These external circulatingsystems add considerably to the cost of using these types ofshell-and-tube bioreactors.

[0010] What is needed is a shell-and-tube type apparatus to growsuspension and adherent cells that does not require an externalcirculating system for the nutrient media fluid.

BRIEF SUMMARY OF THE INVENTION

[0011] The present invention describes an apparatus and method forgrowing cells. An extra-capillary space is formed between hollow fibersand an enclosed chamber. Cells are placed in the extra-capillary spaceto grow. A media reservoir holds cell-culture media. The cell-culturemedia is allowed to pass through a lumen of the hollow fibers and topass nutrients through the walls of the hollow fibers to the cells inthe extra-capillary space. Flow through the hollow fibers is produced byaction of gravity when a rocking motion is imparted to the mediareservoir or by action of gravity when the media reservoir is rotatedabout a horizontal axis.

[0012] Preferably, the hollow fibers are made of a semi-permeablematerial. The semi-permeable material allows: (1) nutrients and (2)metabolizing gasses to pass from the cell-culture media through thewalls of the hollow fibers to the cells in the extra-capillary space,and (3) cell waste products and (4) gaseous waste products to pass fromthe extra-capillary space through the walls of the hollow fibers to thecell-culture media, while retaining the cells and large secretedproducts within the extra-capillary space.

[0013] Preferably, the media reservoir further includes a membranepermitting gas exchange between an exterior and an interior of the mediareservoir.

[0014] Preferably, the media reservoir includes a first opening foraccessing the interior of the media reservoir. The first opening allows:(1) fresh cell-culture media to be supplied to the media reservoir, (2)stale cell-culture media to be removed from the media reservoir, and (3)cell waste products to be removed from the media reservoir.

[0015] In one embodiment, the enclosed chamber is disposed within themedia reservoir. An extra-chamber space is defined between the mediareservoir and the enclosed chamber. Each hollow fiber has, at each end,a lumen open to the extra-chamber space. Preferably, the media reservoirincludes a second opening for accessing the extra-capillary space. Thesecond opening allows: (1) developing cells to be placed into theextra-capillary space, (2) mature cells to be removed from theextra-capillary space, (3) secreted products to be harvested, and (4)the cells to be treated with reagents, drugs, and/or DNA or RNA vectors.

[0016] In another embodiment, the media reservoir is configured to causea flow of the cell-culture media between a first port and a second port.The enclosed chamber is connected between the first and second ports.Each hollow fiber has, at each end, a lumen open to the flow of thecell-culture media between the first and second ports. Preferably, theenclosed chamber includes a second opening for accessing theextra-capillary space. The second opening allows: (1) developing cellsto be placed into the extra-capillary space, and (2) mature cells to beremoved from the extra-capillary space.

[0017] Further features and advantages of the invention as well as thestructure and operation of various embodiments of the present inventionare described in detail below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0018] The accompanying drawings, which are incorporated herein and formpart of the specification, illustrate the present invention and,together with the description, further serve to explain the principlesof the invention and to enable a person skilled in the pertinent art tomake and use the invention.

[0019]FIG. 1 is a cutaway, cross sectional side view of a preferredembodiment of the apparatus of the present invention.

[0020]FIG. 2 is a cutaway end view showing a partial cross section ofthe embodiment shown in FIG. 1 taken along section A-A′.

[0021]FIG. 3 is similar to FIG. 1, but illustrates how a rocking motioncauses the flow of the cell-culture media.

[0022]FIG. 4 is a side view of an alternative preferred embodiment ofthe apparatus of the present invention.

[0023]FIG. 5 shows a top view of the embodiment shown in FIG. 4.

[0024]FIG. 6 is a flowchart illustrating the method 600 of the presentinvention.

[0025] The preferred embodiments of the invention are described withreference to the figures where like reference numbers indicate identicalor functionally similar elements. Also in the figures, the left mostdigit of each reference number identifies the figure in which thereference number is first used.

DETAILED DESCRIPTION OF THE INVENTION

[0026]FIG. 1 is a cutaway, cross sectional side view of a preferredembodiment of the apparatus of the present invention. In FIG. 1, a mediareservoir 102 holds cell-culture media 104 and is configured to berocked or rotated about a horizontal axis of rotation 106 that extendsinto the drawing sheet of FIG. 1. An enclosed chamber 108 is disposedwithin media reservoir 102, wherein an extra-chamber space 110 isdefined between media reservoir 102 and enclosed chamber 108.

[0027] A plurality of hollow fibers 112 pass through enclosed chamber108 and are secured at each end by a first potting structure 114 and asecond potting structure 116. An extra-capillary space 118 is definedbetween an interior of enclosed chamber 108 and the exterior surfaces ofthe hollow fibers 112. The hollow fibers 112 are oriented substantiallyparallel to a longitudinal axis 120, which is substantiallyperpendicular to horizontal axis of rotation 106.

[0028] In a representative embodiment of the invention, 25 to 200 hollowfibers 112 are disposed within enclosed chamber 108. The length of thehollow fibers 112 is from about 3 to 10 centimeters, and the diameter isfrom about 200 to 1,000 microns. Preferably, hollow fibers 112 have alength-to-diameter ratio of less than about 170:1 to reduce the headloss of cell-culture media 104 that passes through them. Because thepresent invention relies on action of gravity to cause the flow ofcell-culture media 104, it is preferred that the hollow fibers 112 havea length-to-diameter ratio small enough to allow a sufficient rate offlow through them. Cumulatively, hollow fibers 112 can support acell-culture media flow rate from about 5 to 100 milliliters per minuteand have a surface area from about 25 to 1,000 square centimeters.However, one skilled in the art would recognize embodiments of thepresent invention with both a greater and a fewer number of hollowfibers 112 (including as few as one hollow fiber 112) and with differentparameters defining them.

[0029]FIG. 2 is a cutaway end view showing a partial cross section ofthe embodiment shown in FIG. 1 taken along section A-A′. In FIG. 2, eachhollow fiber 112 has, at each end, a lumen 202 open to extra-chamberspace 110 such that cell-culture media 104 can pass through the lumen202 of hollow fiber 112 and pass nutrients through the walls of hollowfiber 112 to nourish the cells in the extra-capillary space 118.

[0030] Preferably, each hollow fiber 112 is made of a semi-permeablematerial. The semi-permeable material allows: (1) nutrients and (2)metabolizing gasses to pass from the cell-culture media 104 through thewalls of the hollow fibers 112 to the cells in the extra-capillary space118, and (3) cell waste products and (4) gaseous waste products to passfrom extra-capillary space 118 through the walls of the hollow fibers112 to cell-culture media 104, while retaining the cells and largesecreted products within extra-capillary space 118. Consumption ofnutrients and metabolizing gasses by the cells establishes a gradientbetween extra-chamber space 110 and extra-capillary space 118. Thegradient causes nutrients and gasses to diffuse through the walls of thehollow fibers 112 and into extra-capillary space 118. Conversely, abuild up of cell waste products and gaseous waste products in theextra-capillary space 118 establishes a reverse gradient betweenextra-capillary space 118 and extra-chamber space 110. This reversegradient causes these waste products to diffuse through the walls of thehollow fibers 112 into the lumens 202 and finally into extra-chamberspace 110.

[0031] Preferably, in one embodiment, the semi-permeable material haspores with diameters no larger than 0.2 microns. In various embodiments,the semi-permeable material can be made of one or more of polysulfone,modified polysulfone, polyvinyledine fluoride, cellulose acetate,acrylic copolymer, and a cellulose derivative, wherein said cellulosederivative is one or more of a mixed ester of cellulose andcupra-ammonium rayon. However, one skilled in the art will recognizethat other materials can be used for the semi-permeable material.

[0032] Returning to FIG. 1, media reservoir 102 preferably includes anopening 122 for accessing extra-chamber space 110. Opening 122 allows:(1) fresh cell-culture media to be supplied to media reservoir 102, (2)stale cell-culture media to be removed from media reservoir 102, and (3)cell waste products to be removed from media reservoir 102. A lid 124 isused to seal opening 122.

[0033] Preferably, media reservoir 102 includes an opening 126 foraccessing extra-capillary space 118. In an embodiment, opening 126includes a port 128 passing through extra-chamber space 110 to provideaccess to extra-capillary space 118. Opening 126 allows: (1) developingcells to be placed into extra-capillary space 118, (2) mature cells tobe removed from extra-capillary space 118, (3) secreted products to beharvested, and (4) the cells to be treated with reagents, drugs, and/orDNA or RNA vectors. In an embodiment, media reservoir 102 includes morethan one opening 126.

[0034] Preferably, media reservoir 102 further includes a gas permeablemembrane 130 permitting gas exchange between an environment exterior tomedia reservoir 102 and extra-chamber space 110. As discussed above,gasses are exchanged between extra-chamber space 110 and extra-capillaryspace 118 through the walls of hollow fibers 112. Membrane 130 permitsthe exchange of the waste gasses from extra-chamber space 110 with freshgasses from the environment exterior to media reservoir 102. Transversemembers 134 provide support and structural integrity to media reservoir102 along a face that includes membrane 130. In one embodiment, membrane130 is made of silicone. However, one skilled in the art will recognizethat other materials can be used for the membrane.

[0035] A dam 136 is disposed in media reservoir 102 to impede flow ofcell-culture media 104 in extra-chamber space 110 when media reservoir102 is rocked or rotated about horizontal axis of rotation 106. Dam 136also serves to encourage flow of cell-culture media 104 through thehollow fibers 112. In an embodiment in which enclosed chamber 108 spansthe width of media reservoir 102 along horizontal axis of rotation 106,enclosed chamber 108 and dam 136 are integrated.

[0036]FIG. 3 is similar to FIG. 1, but illustrates how a rocking orrotating motion causes the flow of cell-culture media. In FIG. 3, oneskilled in the art will recognize how dam 136, by impeding the flow ofcell-culture media 104 in extra-chamber space 110, simultaneouslyincreases the static head pressure of a raised portion 302 ofcell-culture media 104 and decreases the static head pressure of alowered portion 304 of cell-culture media 104 that would otherwise existin the absence of dam 134. Thus, by increasing the differential pressureacross the hollow fibers 112, dam 136 serves to encourage flow ofcell-culture media 104 through the hollow fibers 112.

[0037]FIG. 4 is a side view of an alternative preferred embodiment ofthe apparatus of the present invention. FIG. 5 shows a top view of theembodiment shown in FIG. 4. In FIGS. 4 and 5, a media reservoir 402holds cell-culture media 104 and is configured to cause a flow ofcell-culture media 104 between a first port 502 and a second port 504 inresponse to a rocking or rotating motion imparted to media reservoir402. An enclosed chamber 404 is connected between first port 502 andsecond port 504. One skilled in the art will recognize that theseconnections can be realized by several means including, but not limitedto, a first tubing section 506 connected between first port 502 andenclosed chamber 404, and a second tubing section 508 connected betweensecond port 504 and enclosed chamber 404.

[0038] A plurality of hollow fibers 510 passes through enclosed chamber404 and is secured at each end by a first potting structure 512 and asecond potting structure 514. An extra-capillary space 516 is definedbetween an interior of the enclosed chamber 404 and the exteriorsurfaces of the hollow fibers 510. Each hollow fiber 510 has, at eachend, a lumen open to the flow of cell-culture media 104 between firstport 502 and second port 504 such that cell-culture media 104 can passthrough the lumen of hollow fiber 510 and pass nutrients through thewalls of hollow fiber 510 to nourish the cells in extra-capillary space516. Other functions and parameters of the hollow fibers 510 areidentical to those included in the first preferred embodiment describedabove.

[0039] In a representative embodiment of the invention, 25 to 200 hollowfibers 510 are disposed within enclosed chamber 404. The length of thehollow fibers 510 is from about 3 to 10 centimeters, and the diameter isfrom about 200 to 1,000 microns. Preferably, hollow fibers 510 have alength-to-diameter ratio of less than about 170:1 to reduce the headloss of cell-culture media 104 that passes through them. Because thepresent invention relies on action of gravity to cause the flow ofcell-culture media 104, it is preferred that the hollow fibers 510 havea length-to-diameter ratio small enough to allow a sufficient rate offlow through them. Cumulatively, hollow fibers 510 can support acell-culture media flow rate from about 5 to 100 milliliters per minuteand have a surface area from about 25 to 1,000 square centimeters.However, one skilled in the art would recognize embodiments of thepresent invention with both a greater and a fewer number of hollowfibers 510 (including as few as one hollow fiber 510) and with differentparameters defining them.

[0040] Enclosed chamber 404 can be a standard, commercially availableshell-and-tube type bioreactor designed for use with an externalcirculating system.

[0041] Preferably, media reservoir 402 comprises a tray 406, a first bag408 connected to first port 502, and a second bag 410 connected tosecond port 504. This configuration serves to encourage flow ofcell-culture media 104 through the hollow fibers 510 when a rocking orrotating motion is imparted to media reservoir 402. Advantageously, thisconfiguration allows for flexibility in the orientation of hollow fibers510 with respect to horizontal axis of rotation 106. However, oneskilled in the art will recognize other embodiments by which mediareservoir 402 can be realized. These include, but are not limited to, aconfiguration using one bag connected to both ports, wherein the bagincludes a damming or dividing mechanism to encourage flow ofcell-culture media 104 through enclosed chamber 404 by increasing thedifferential pressure between the two ports when the bag is rocked orrotated.

[0042] In an embodiment, first bag 408 and second bag 410 are made of agas permeable material such as silicone permitting gas exchange betweenthe environment exterior to media reservoir 102 and an interior 414 ofmedia reservoir 402. In an alternate embodiment, only a portion of bags408, 410 are made from a gas permeable material. Similar to the processdiscussed above, gasses are exchanged between an interior 412 of mediareservoir 402 and extra-capillary space 516 through the walls of hollowfibers 510. The gas permeable material permits the exchange of the wastegasses from the interior 412 of media reservoir 402 with fresh gassesfrom the environment exterior to media reservoir 402.

[0043] Preferably, media reservoir 402 includes an opening 518 foraccessing interior 412 of media reservoir 402. As described above,opening 518 allows: (1) fresh cell-culture media to be supplied to mediareservoir 402, (2) stale cell-culture media to be removed from mediareservoir 402, and (3) cell waste products to be removed from mediareservoir 402. A lid 414 is used to seal opening 518. Where mediareservoir 402 comprises first bag 408 and second bag 410, each bag hasits own opening 518.

[0044] Preferably, enclosed chamber 404 includes an opening 520 foraccessing extra-capillary space 516. As described above, opening 520allows: (1) developing cells to be placed into extra-capillary space516, (2) mature cells to be removed from extra-capillary space 516, (3)secreted products to be harvested, and (4) the cells to be treated withreagents, drugs, and/or DNA or RNA vectors. In an embodiment, enclosedchamber 404 includes more than one opening 520.

[0045] In an embodiment, enclosed chamber 404 is attached to tray 406 bya clip 416. However, one skilled in the art would recognize other meansby which enclosed chamber 404 could be held in an appropriate positionwith respect to media reservoir 402.

[0046]FIG. 6 is a flowchart illustrating the method 600 of the presentinvention. In FIG. 6, at a step 602, the media reservoir is accessed tosupply cell-culture media. Where cell-culture media has previously beensupplied to the media reservoir, one skilled in the art will recognizethat step 602 can be omitted. At a step 604, cells are placed into theextra-capillary space formed between the semipermeable hollow fibers andthe enclosed chamber. At a step 606, the media reservoir is rocked backand forth to cause a flow of the cell-culture media to pass through thelumens of the hollow fibers by action of gravity. Alternatively, at step606, the media reservoir can be rotated about a horizontal axis ofrotation to cause a flow of the cell-culture media to pass through thelumens of the hollow fibers by action of gravity. The cell-culture mediapass nutrients through the walls of the hollow fibers to nourish thecells in the extra-capillary space. In an embodiment, the hollow fibersare oriented substantially perpendicular to the horizontal axis ofrotation.

[0047] Optionally, at a step 608, the extra-capillary space is accessedto remove the cells. Optionally, at a step 610, the media reservoir isaccessed to remove the cell-culture media and any waste products.

CONCLUSION

[0048] While various embodiments of the present invention have beendescribed above, it should be understood that they have been presentedby way of example, and not limitation. It will be apparent to personsskilled in the relevant art that various changes in form and detail canbe made therein without departing from the spirit and scope of theinvention. Thus the present invention should not be limited by any ofthe above-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

What is claimed is:
 1. An apparatus for growing cells, comprising: a media reservoir for holding cell-culture media and configured to be rocked or rotated about a horizontal axis of rotation; an enclosed chamber disposed within said media reservoir, wherein an extra-chamber space is defined between said media reservoir and said enclosed chamber; and at least one hollow fiber passing through said enclosed chamber, wherein each said at least one hollow fiber is substantially oriented upon a longitudinal axis substantially perpendicular to said horizontal axis of rotation, wherein each said at least one hollow fiber has, at each end, a lumen open to said extra-chamber space, and wherein an extra-capillary space is defined between an interior of said enclosed chamber and an exterior surface of said at least one hollow fiber.
 2. The apparatus of claim 1, further comprising: a first opening in said media reservoir for accessing said extra-chamber space; and a second opening in said media reservoir for accessing said extra-capillary space.
 3. The apparatus of claim 1, wherein said at least one hollow fiber comprises a plurality of hollow fibers.
 4. The apparatus of claim 3, wherein each of said plurality of hollow fibers is made of a semi-permeable material.
 5. The apparatus of claim 4, wherein said semi-permeable material is one or more of polysulfone, modified polysulfone, polyvinyledine fluoride, cellulose acetate, acrylic copolymer, and a cellulose derivative, wherein said cellulose derivative is one or more of a mixed ester of cellulose and cupra-ammonium rayon.
 6. The apparatus of claim 5, wherein said semi-permeable material has pores with diameters no larger than 0.2 μm.
 7. The apparatus of claim 1, wherein said media reservoir comprises a membrane permitting gas exchange between an exterior of said media reservoir and said extra-chamber space.
 8. The apparatus of claim 7, wherein said membrane is made of silicone.
 9. The apparatus of claim 1, further comprising: a dam disposed in said media reservoir to impede flow of said media in said extra-chamber space when said media reservoir is rocked or rotated about said horizontal axis of rotation and to encourage flow of said media through said at least one hollow fiber.
 10. A method for growing cells, comprising: placing cells into an extra-capillary space formed between at least one semi-permeable hollow fiber and an enclosed chamber; and rocking a media reservoir back and forth to cause a flow of a cell-culture media to pass through a lumen of the at least one hollow fiber by action of gravity, whereby the cell-culture media passes nutrients through walls of the at least one hollow fiber to nourish the cells in the extra-capillary space.
 11. The method of claim 10, wherein said rocking step comprises: rotating a media about a horizontal axis to cause a flow of a cell-culture media to pass through a lumen of the at least one hollow fiber by action of gravity, whereby the cell-culture media passes nutrients through walls of the at least one hollow fiber to nourish the cells in the extra-capillary space.
 12. The method of claim 10, wherein the at least one hollow fiber is oriented substantially perpendicular to a horizontal axis of rotation.
 13. The method of claim 10, further comprising: accessing the extra-capillary space to remove the cells.
 14. The method of claim 10, further comprising: accessing the extra-capillary space to harvest secreted products.
 15. The method of claim 10, further comprising: accessing the extra-capillary space to treat the cells with one or more of reagents, drugs, DNA vectors, and RNA vectors. 